Amphiphilic block copolymers and nano particles comprising the same

ABSTRACT

An amphiphilic block copolymer. The amphiphilic block copolymer includes one or more hydrophilic polymers, one or more hydrophobic polymer, and one or more zwitterions. The invention also provides a nano particle and carrier comprising the amhpiphilic block copolymer for delivery of water-insoluble drugs, growth factors, genes, or cosmetic substances.

BACKGROUND

The invention relates to a polymer, and more specifically to abio-compatible and bio-degradable block copolymer and a nano particlecomprising the same.

To improve curative effects and reduce side effects, an optimal drugdelivery carrier which can precisely target at tumor cell and producehigh drug concentration around the focus is required. Such carriermaterials must be bio-compatible, bio-degradable, and be invisible toimmune system detection to avoid macrophage attack. Common carriermaterials comprise block copolymers. Some, however, lackbio-degradability, long-term stability, and invisibility to the immunecells in blood circulation.

Sugiyama provides a zwitterionic copolymer comprising a hydrophilicchain of 2-(methacryloyloxy)ethyl phosphorylcholine (MPC) and ahydrophobic chain ends of cholesteryl with critical micelleconcentration (CMC) of 2.5×10⁻⁴−2.7×10⁻⁵ wt % recited in Journal ofPolymer Science Part A: Polymer Chemistry (2003) 1992-2000. Suchmaterial forms a complex with cholesterol molecules in condensed layermorphology with a 3.52 nm interval therebetween. This copolymer provideshemo-compatibility and encapsulates water-insoluble drugs by hydrophobiccholesterol groups attached on the polymer chains. The material,however, lacks bio-degradability.

Stenzel provides a zwitterionic block copolymer comprisingpoly(2-acryloyloxyethyl phosphorylcholine) and poly(butyl acrylate) withbio-compatibility formed by reversible addition fragmentation transfer(RAFT) recited in Macromolecular Bioscience (2004) 445-453. The nanoparticle formed in water and methanol binary solvent thereby has adiameter of about 100-180 nm. This copolymer, however, also lacksbio-degradability.

Nakabayashi provides a bio-degradable poly(L-lactic acid) (PLLA)material comprising phosphorylcholine such asL-α-glycerophosphorylcholine (LGPC) recited in Journal of BiomedicalMaterials Research part A (2003) 164-169. Various molecular weight PLLAmaterials can be polymerized using LGPC as an initiator. According to ablood cell experiment, as the phosphorylcholine content of PLLAincreases, blood-cell adsorption reduces. This material, however, lacksmicelle characteristics.

Oishi provides a novel zwitterionic poly(fumaramate) material comprisingphosphorylcholine with CMC of about 1.7×10⁻³−1.0×10⁻³M recited inPolymer (1997) 3109-3115. The CMC is altered by conducting isopropyl andmethyl groups to fumaramate. According to a bovine serum albuminadsorption experiment, as the phosphorylcholine content ofpoly(fumaramate) increases, bovine serum albumin adsorption is reduced.

Sommerdijk provides a polystyrene material comprising tetra(ethyleneglycol) and phosphorylcholine bonded to its side chain withbio-compatibility recited in Journal of Polymer Science Part A: PolymerChemistry (2001) 468-474. According protein adsorption experiment andin-vitro human dermal fibroblasts culture, only polystyrene materialcontaining tetra(ethylene glycol) and phosphorylcholine can reduceprotein adsorption and fibroblasts growth, as compared with polystyrenebonded with tetra(ethylene glycol) or phosphorylcholine alone.

Langer provides a bio-degradable injection-type nano particle comprisingdiblock (AB) or triblock (ABC) copolymer disclosed in U.S. Pat. Nos.5,543,158 and 6,007,845. A is a hydrophilic chain such as poly(alkyleneglycol), B is a hydrophobic chain such as PLGA, and C is a bio-activesubstance such as antibody. The particle can target at cells or organsby the bio-active substance bonded thereto. These applications, however,fail to disclose AB or ABC can provide invisibility to bio-recognition.

Kim provides a bio-degradable drug-contained micelle comprising diblock(BA) or triblock (ABA or BAB) copolymer with a preferable molecularweight of about 1430-6000 and a hydrophilic chain ratio of 50-70 wt %disclosed in U.S. Pat. No. 6,322,805. A is a bio-degradable hydrophobicchain and B is a hydrophilic chain such as PEG. Drugs such as paclitaxelare encapsulated by physical manner. The application, however, fails todisclose ABC-type triblock copolymer.

Marchant provides an anti-thrombosis triblock copolymer comprising ahydrophobic bio-degradable polymer and a hydrophilic polysaccharidechain such as dextran or heparin disclosed in U.S. Pat. No. 5,741,852.

Ishihara provides a method for preparing a phosphorylcholine-containedpolymer in aqueous phase disclosed in U.S. Pat. No. 6,204,324.Polymerizable monomers, phosphorylcholine, are polymerized by addinginitiator in aqueous medium. The results are then purified by aseparation film, containing impurity less than 2000 ppm.

SUMMARY

The invention provides a block copolymer comprising one or morehydrophobic blocks and one or more zwitterions. The block copolymerfurther comprises one or more hydrophilic blocks bonded to thehydrophobic block.

The invention also provides a nano particle comprising one or more thedisclosed block copolymers.

The invention further provides a nano carrier comprising the disclosednano particle and an active substance encapsulated inside the particle.

A detailed description is given in the following embodiments withreference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the invention can be more fully understood by reading thesubsequent detailed description and examples with references made to theaccompanying drawings, wherein:

FIG. 1 shows ¹H-NMR spectrum of PEG-PCL-PC.

FIG. 2 shows ¹H-NMR spectrum of PEG-PCL-NS.

FIG. 3 shows ¹H-NMR spectrum of PEG-PCL-benzyl histidine.

FIGS. 4-6 shows mean fluorescence intensity of DCF in variousconditions.

DETAILED DESCRIPTION

The invention provides a block copolymer comprising one or morehydrophobic polymers and one or more zwitterions.

The copolymer is an amphiphilic block copolymer. The block copolymercomprises diblock copolymer, with CMC of about 0.1-0.01 wt %. Thehydrophobic block has a molecular weight of about 1000-30000 and maycomprise polyester such as polycaprolactone (PCL), polyvalerolactone(PVL), poly(lactide-co-glycolide) (PLGA), polylactic acid (PLA),polybutyrolactone (PBL), polyglycolide, and polypropiolactone (PPL). Thezwitterions may comprise phosphorylcholine (PC), sulfobetaine (NS), oramino acids.

The block copolymer further comprises one or more hydrophilic blocksbonded to the hydrophobic block to form a triblock copolymer ofhydrophilic block-hydrophobic block-zwitterion. The hydrophilic blockhas a molecular weight of about 550-20000 and may comprise polyethyleneglycol (PEG), hyaluronic acid (HA), or poly-γ-glutamic acid (γ-PGA). Theblock copolymer comprising diblock or triblock is bio-degradable andbio-compatible.

The invention also provides a nano particle comprising one or more thedisclosed block copolymers.

The nano particle has a hydrophobic interior and hydrophilic surface andits diameter is about 20-1000 nm.

Due to the chain flexibility of hydrophilic block in blood, zwitterionscapable of keeping particle structure without recognized by immune cellsduring drug releasing period, and a hydrophobic block decomposable byenzyme or hydrolysis, the novel biomedical nano particle providesbio-compatibility, bio-degradability, and invisibility to immune cells.After the hydrophobic block is decomposed, remaining harmless substancessuch as hydrophilic block and zwitterion are dissolved in blood and thenremoved from renal system.

The invention further provides a nano carrier comprising the disclosednano particle and an active substance encapsulated inside the particle.

The active substance may comprise water-insoluble drugs such ascamptothecin or derivatives thereof, growth factors, genes, orwater-insoluble cosmetic substances such as ingredients for skincare.The nano carrier can be delivered by oral, transdermal administration,injection, or inhalation.

The preparation of the biomedical polymer is described as follows.First, a copolymer comprising a hydrophilic block and a hydrophobicblock, such as PEG-PCL, PEG-PVL, and PEG-PPL, is prepared. Next, thecopolymer is dissolved in a solvent, such as dichloromethane (CH₂Cl₂),and its terminal is added with a chemical group, such as2-chloro-2-oxo-1,3,2-dioxaphospholane (COP), succinic anhydride (SA),and 3-(dimethylamino)propylamine (DMAPA), to form a modified copolymer.After the modified copolymer is dissolved in a solvent such asacetonitrile and chloroform, its modified terminal is reacted withanother chemical group, such as trimethylamine (TMA), 1,3-propanesultone (PS), and benzyl histidine, to form zwitterions. Thus, acopolymer comprising a hydrophilic block, a hydrophobic block, andzwitterions is obtained.

EXAMPLES Synthesis of PEG-PCL-PC

(1) Polymerization of PEG-PCL:

60 g poly(ethylene glycol) (PEG, 5000 g/mole) and 12 g ε-caprolactonewere added to a 250 ml glass reactor (8 cm×8 cm×10 cm) with gradualheating until totally dissolved. The PEG-PCL polymerization then beganby adding 0.38 ml catalyst of stannous 2-ethylhexanoate at 160° C. for24 hours. The crude product was purified by dissolving indichloromethane and re-precipitating in diethyl ether. The purificationwas repeated three times and vacuum-dried at 40° C. for 24 hours. Thus,a PEG-PCL copolymer was obtained.

(2) Synthesis of PEG-PCL-COP:

5 g PEG-PCL copolymer and 0.43 g triethylamine (TEA) were dissolved in70 ml dichloromethane at 0° C. with mechanical stirring in a 250 mlflask. Another solution containing 3.5 g2-chloro-2-oxo-1,3,2-dioxaphospholane (COP) dissolved in 30 mldichloromethane was prepared simultaneously. The COP-contained solutionwas then gradually dropped into the flask within 1 hour and reacted at0° C. for 4 hours. The resulting solution was then warmed to roomtemperature and filtered by 0.45 μm filter paper to remove theby-product, triethylammonium chloride. After removing dichloromethane, amodified copolymer, PEG-PCL-COP, was obtained.

(3) Synthesis of PEG-PCL-PC:

PEG-PCL-COP was dissolved in 70 ml acetonitrile at room temperature in aflask. 10 ml trimethylamine (33% in ethanol) was then dropped into theflask and reacted at 60° C. for 24 hours with stirring. After removingthe solvent, the resulting solution was extracted three times bydichloromethane/water. After removing dichloromethane and vacuum-dryingfor 24 hours, white solid resulted, PEG-PCL-PC copolymer, were obtained.Its ¹H-NMR is shown in FIG. 1.

Synthesis of PEG-PVL-PC

(1) Polymerization of PEG-PVL:

60 g poly(ethylene glycol) (PEG, 5000 g/mole) and 12 g δ-valerolactonewere added to a 250 ml glass reactor (8 cm×8 cm×10 cm) with gradualheating until totally dissolved. The PEG-PVL polymerization then beganby adding 0.38 ml catalyst of stannous 2-ethylhexanoate at 160° C. for 8hours. The result was dissolved in dichloromethane and re-precipitatedby adding diethyl ether. The white precipitate was then washed threetimes and vacuum-dried at 40° C. for 24 hours. Thus, a PEG-PVL copolymerwas obtained.

(2) Synthesis of PEG-PVL-COP:

5 g PEG-PVL copolymer and 0.43 g triethylamine (TEA) were dissolved in70 ml dichloromethane at 0° C. with mechanical stirring in a 250 mlflask. Another solution containing 3.5 g2-chloro-2-oxo-1,3,2-dioxaphospholane (COP) dissolved in 30 mldichloromethane was prepared simultaneously. The COP-contained solutionwas then gradually dropped into the flask within 1 hour and reacted at0° C. for 6 hours. The resulting solution was then warmed to roomtemperature and filtered by 0.45 μm filter paper to remove theby-product, triethylammonium chloride. After removing dichloromethane, amodified copolymer, PEG-PVL-COP, was obtained.

(3) Synthesis of PEG-PVL-PC:

PEG-PVL-COP was dissolved in 70 ml acetonitrile at room temperature in aflask. 10 ml trimethylamine (33% in ethanol) was then dropped into theflask and reacted at 60° C. for 24 hours with stirring. After removingthe solvent, the resulting solution was extracted three times bydichloromethane/water. After removing dichloromethane and vacuum-dryingfor 24 hours, white solid results, PEG-PVL-PC copolymer, were obtained.

Synthesis of PEG-PCL-NS

(1) Synthesis of PEG-PCL-SA:

3 g PEG-PCL copolymer and 0.1 g 4-dimethylaminopyridine (DMAP) weredissolved in 60 ml dichloromethane at 0° C. with mechanical stirring ina 250 ml flask. Another solution containing 0.1 g triethylamine (TEA)and 0.5 g succinic anhydride (SA) dissolved in 10 ml dichloromethane wasprepared simultaneously. The SA-contained solution was then graduallydropped into the flask within 1 hour and reacted at 25° C. for 24 hours.The resulting solution was then precipitated three times by addingdiethyl ether and vacuum-dried for 24 hours. Thus, a modified copolymer,PEG-PCL-SA, was obtained.

(2) Synthesis of PEG-PCL-TA:

2 g PEG-PCL-SA copolymer, 0.2 g 1,3-dicyclohexylcarbodiimide (DCC), and0.1 g N-hydroxysuccinimide (NHS) were dissolved in 40 ml dichloromethaneat 0° C. with mechanical stirring in a 250 ml flask. Another solutioncontaining 0.1 g 3-(dimethylamino)propylamine (DMAPA) dissolved in 10 mldichloromethane was prepared simultaneously. The DMAPA-containedsolution was then gradually dropped into the flask within 1 hour andreacted at 25° C. for 24 hours. The resulting solution was thenprecipitated three times by adding diethyl ether and vacuum-dried for 24hours. Thus, a modified copolymer, PEG-PCL-TA, was obtained.

(3) Synthesis of PEG-PCL-NS:

0.15 g PEG-PCL-TA was dissolved in 60 ml chloroform at 0° C. in a flask.0.36 g 1,3-propane sultone (20% in chloroform) was then dropped into theflask and reacted at 30° C. for 24 hours with stirring. The resultingsolution was then precipitated three times by adding diethyl ether andvacuum-dried for 24 hours. Thus, a copolymer, PEG-PCL-NS, was obtained.Its ¹H-NMR is shown in FIG. 2.Synthesis of PEG-PCL-Benzyl HistidineCoupling of PEG-PCL-SA and Benzyl Histidine

2 g PEG-PCL-SA copolymer, 0.1547 g N,N-dicyclohexyl carbodiimide (DCC)and 0.0863 g N-hydroxysuccinimide (NHS) were dissolved in 50 mldichloromethane at 40° C. with mechanical stirring in a 250 ml flask.Another solution containing 0.368 g im-benzyl-L-histidine (His(Bzl))dissolved in 100 ml methanol was prepared simultaneously. TheHis(Bzl)-contained solution was then added into the flask and reacted at60° C. for 24 hours. The resulting solution was then cooled down to roomtemperature and filtered by 0.45 μm filter paper to remove theun-reaction material, His(Bzl). After removing dichloromethane, amodified copolymer, PEG-PCL-His(Bzl), was obtained. Its ¹H-NMR is shownin FIG. 3.

The Critical Micelle Concentration (CMC) of Polymeric Micelles

The micelle CMC is measured by the method recited in Jeong et al., 1999.0.4 mM DPH (1,6-diphenyl-1,3,5-hexatriene) water solution and copolymerswith 2−2×10⁻⁴ wt % were mixed. Absorption in 356 nm of the watersolution was then measured by a UV-Vis spectrometer. Finally, theabsorption was plotted in a diagram against logarithm of polymerconcentrations. A turning point formed by two various slopes in thediagram is CMC.

Table 1 recited CMC values of the block copolymers is shown here.

TABLE 1 Molecular Molecular weight of PEG weight of PCL CMC Sample ID(g/mole) (g/mole) (×10⁻² wt %) PEG-PCL-PC1 5000 1900 3.26 PEG-PCL-PC25000 1100 17.92 PEG-PCL-NS1 2000 1000 1.46 PEG-PCL-NS2 2000 2000 4.47PEG-PCL-NS3 5000 2500 3.95 PEG-PCL-NS4 5000 3700 7.76Preparation of Micelles and Analysis of Sizes Thereof

10 mg polymer was dissolved in 1 ml THF to form a solution. The solutionwas gradually dropped into 30 ml deionized water by a 2.5 ml syringe andstirred. The solution was then placed in a dialysis membrane to dialyzefor 24 hours to form a micelle solution. Finally, 3-5 ml micellesolution was placed in an acrylic cuvette to measure micelle sizes andtheir distribution by a photon correlation spectroscopy (MalvernInstrument Zetasizer Nano ZS), as shown in Table 2.

TABLE 2 Molecular Molecular Hydrodynamic weight of PEG weight of PCLdiameter Sample ID (g/mole) (g/mole) (nm) PEG-PCL-PC1 5000 1900 113.8PEG-PCL-PC2 5000 1100 — PEG-PCL-NS1 2000 1000 148.3 PEG-PCL-NS2 20002000 34.7 PEG-PCL-NS3 5000 2500 27.2 PEG-PCL-NS4 5000 3700 —In-Vitro Stealth Test

In blood, macrophage may be activated and produce reactive oxygenspecies after foreign substance is recognized thereby. Thus, theinvisibility of micelles to macrophage can be determined by measuringthe reactive oxygen species contents. 2′,7′-dichlorodihydrofluoresceindiacetate (DCFDA) was added into a microphage culture (RAW 264.7). Amicelle solution having concentration above CMC was then added andcultivated for 24 hours. If micelles activate macrophage, DCFDA may beconverted into fluorescent DCF (2′,7′-Dichlorofluorescin) by reactiveoxygen species and its fluorescence intensity is directly proportionalto reactive oxygen species contents. Finally, the invisibility ofmicelles was obtained by measurement with a flow cytometer. Thefluorescence intensity of DCF in various conditions is shown in FIGS.4-6.

FIG. 4 shows a mean fluorescence intensity of 9.39 without addition ofmicelles. FIG. 5 shows a mean fluorescence intensity of 10.8 withaddition of micelles (PEG-PCL-PC2). FIG. 6 shows a mean fluorescenceintensity of 20.13 with addition of 1 μm PMA for 24-hour activation. Dueto the mean fluorescence intensity between (a) and (b) are similar, wecan assert that the micelles are invisible.

Preparation of Polymeric Nano Particle

10 mg E50C19-PC was added into 1 mL dimethyl sulfoxide (DMSO) andstirred. After DMSO was removed by freeze-drying, 1 ml and 10% sucrosewas added to hydrate. The freeze-dried solids were then dissolved toform a suspension. After ultra-sonicating for 10 min, polymer nanoparticles were formed. The particle size distribution of 120-150 nm wasobtained by a laser particle size analyzer (Coulter N4 plus).

Preparation of Nano Carrier Containing Drugs

Camptothecin is a water-insoluble drug and has solubility of only 3μg/ml. 1 mg camptothecin and 10 mg E50C19-PC were added into 1 mldimethyl sulfoxide (DMSO) and stirred. After DMSO was removed byfreeze-drying, 1 ml and 10% sucrose was added to hydrate. Thefreeze-dried solids were then dissolved to form a suspension. Afterultra-sonicating for 10 min, polymer nano particles containingcamptothecin were formed. The suspension was then filtered with a 0.45μm filter to remove un-encapsulated camptothecin crystals. The particlesize distribution of 130-190 nm was obtained by a laser particle sizeanalyzer (Coulter N4 plus). The camptothecin concentration of 0.9 mg/mlin a micelle solution was measured by a HPLC. The solubility ofcamptothecin was increased to 300 times.

Hemolysis Test of Nano Carrier Containing Drugs

The toxicity of polymer micelles with and without camptothecin toerythrocyte was quantitatively analyzed by the ASTM F756 standardoperation. The results shown in Table 3 inducate that the nano carriersare non-hymolytic.

TABLE 3 Hemolytic Carrier type index (%) Hemolytic grade PEG-PCL-PC1(placebo) −0.07 Non-hemolytic PEG-PCL-PC1 nano carrier containing 0.44Non-hemolytic camptothecin

While the invention has been described by way of example and in terms ofpreferred embodiment, it is to be understood that the invention is notlimited thereto. To the contrary, it is intended to cover variousmodifications and similar arrangements (as would be apparent to thoseskilled in the art). Therefore, the scope of the appended claims shouldbe accorded the broadest interpretation so as to encompass all suchmodifications and similar arrangements.

What is claimed is:
 1. A block copolymer comprising: a hydrophobicblock, a hydrophilic block bonded to the hydrophobic block, and only onezwitterion, wherein the zwitterion, having one positive charge and onenegative charge, is bonded to a terminal end of the hydrophobic block;the hydrophobic block is polycaprolactone (PCL), polyvalerolactone(PVL), poly(lactide-co-glycolide) (PLGA), polylactic acid (PLA),polybutyrolactone (PBL), polyglycolide, or polypropiolactone (PPL); andthe hydrophilic block is polyethylene glycol (PEG), hyaluronic acid (HA)or poly-γ-glutamic acid (PGA).
 2. The block copolymer as claimed inclaim 1, wherein the hydrophobic block has a molecular weight of about1000-30000.
 3. The block copolymer as claimed in claim 1, wherein thezwitterion comprises phosphorylcholine (PC), sulfobetaine (NS), or anamino acid.
 4. The block copolymer as claimed in claim 1, wherein thehydrophilic block has a molecular weight of about 550-20000.
 5. Theblock copolymer as claimed in claim 1, wherein the copolymer isbio-degradable.
 6. The block copolymer as claimed in claim 1, whereinthe copolymer is bio-compatible.
 7. A nano particle comprising one ormore block copolymers as claimed in claim
 1. 8. The nano particle asclaimed in claim 7, wherein the nano particle has a hydrophobic interiorand hydrophilic surface.
 9. The nano particle as claimed in claim 7,wherein the nano particle has a diameter of about 20-1000 nm.
 10. A nanocarrier, comprising: a nano particle as claimed in claim 7; and anactive substance encapsulated inside the particle.
 11. The nano carrieras claimed in claim 10, wherein the active substance compriseswater-insoluble drugs, growth factors, genes, or water-insolublecosmetic substances.
 12. The nano carrier as claimed in claim 10,wherein the active substance comprises camptothecin or derivativesthereof.
 13. The nano carrier as claimed in claim 10, wherein the activesubstance comprises water-insoluble ingredients for skincare.
 14. Thenano carrier as claimed in claim 10, wherein the nano carrier isdelivered by oral, transdermal administration, injection, or inhalation.15. The block copolymer as claimed in claim 1, wherein the hydrophobicblock is polycaprolactone (PCL), the hydrophilic block is polyethyleneglycol (PEG), and the zwitterion is phosphorylcholine (PC), sulfobetaine(NS), or benzyl histidine.
 16. The block copolymer as claimed in claim15, wherein when the zwitterion is PC, the hydrophilic block has amolecular weight of 5000 and the hydrophobic block has a molecularweight of 1100-1900.
 17. The block copolymer as claimed in claim 15,wherein when the zwitterion is NS, the hydrophilic block has a molecularweight of 2000-5000 and the hydrophobic block has a molecular weight of1000-3700.
 18. The block copolymer as claimed in claim 1, wherein thehydrophobic block is polycaprolactone, the hydrophilic block ispolyethylene glycol, and the zwitterion is phosphorylcholine.
 19. Theblock copolymer as claimed in claim 1, wherein the hydrophobic block ispolylactic acid, the hydrophilic block is polyethylene glycol, and thezwitterion is phosphorylcholine.